Transformation of epithelial cells is closely linked to disturbances of their normal growth/differentiation program. Understanding the balance between epithelial self-renewal and differentiation is thus of great importance, especially since the vast majority of human tumors is of epithelial origin. The main working hypothesis of this proposal is that Notch signaling plays an important role in the control of epithelial self-renewal, differentiation and tumorigenesis. Notch signaling provides a highly conserved mechanism for control of cell fate determination and differentiation in a wide variety of developmental and non-developmental processes. However, whether or not Notch signaling plays an essential function in the balance between epithelial cell growth and differentiation in vertebrates remains to be established. Similarly, although increased Notch expression has been correlated with development of cervical cancer and other keratinocyte-derived tumors, the specific functional consequences of Notch activation on normal keratinocytes, and primary epithelial cells in general, have not been assessed. Mouse keratinocytes will be used as a prototype model system. The following specific aims will be pursued: 1) We will test the hypothesis that Notch activation is required and/or sufficient for the exit of keratinocytes from the cell cycle during differentiation. This hypothesis will be tested with primary keratinocytes under well-defined culture conditions. Notch function will be perturbed by treatment with a Notch ligand-competing peptide as well as adenoviral-mediated expression of sense and anti-sense Notch1 cDNAs. 2) We will test the hypothesis that, besides its role in cell cycle withdrawal, Notch function is required and/or sufficient for other specific aspects of keratinocyte differentiation, such as induction of biochemical markers of differentiation and associated morphological/structural changes. This hypothesis will be tested with primary keratinocytes in culture using the combined approaches outlined above. 3) We will test the hypothesis that Notch function is required for control of keratinocyte growth and differentiation in vivo. This hypothesis will be tested by the analysis of mice with conditional keratinocyte-specific deletions of the Notch1 and Notch2 genes, individually and in combination. The long term consequences of loss of these genes for control of skin homeostasis and tumorigenesis will be investigated by classical chemical carcinogenesis studies as well as in vitro/in vivo assays for keratinocyte Stem cell potential and susceptibility to transformation.